Platelets, also referred to as thrombocytes, are anuclear cell fragments that exist in mammalian blood and mediate blood clot formation and haemostasis. In addition, platelets release growth factors that play a significant role in the repair and regeneration of connective tissues and facilitate wound healing. Platelets are the terminal differentiation product of megakaryocytes (MK), which in turn originate from pluripotent stem cells of the bone marrow. While humoral factors such as thrombopoietin (TPO) were found to influence different cellular steps in megakaryocyte development, the complex maturation, differentiation and localization processes that begin with pluripotent stem cells and end with blood platelets remain incompletely understood.
Platelets have an average lifespan of about 5 to 10 days, and their physiological blood level is normally 150,000 to 450,000/μL. When a patient's levels of circulating platelets are depleted below the physiological range, a condition known as thrombocytopenia can follow. This condition is typically associated with defective formation of haemostatic plugs and bleeding, wherein the risk of bleeding is inversely proportional to the platelet count.
Platelet levels may be lowered by the reduction of platelet productivity in bone marrow, or by platelet consumption, promotion of platelet degradation in periphery, or abnormal platelet distribution. For example, thrombocytopenia can be due to antibody mediated platelet destruction or bone marrow failure from e.g. malignant infiltration or chemotherapy.
Pharmacological intervention is possible in some cases; for example, in certain conditions such as immune mediated thrombocytopenia (wherein the platelets are targeted and destroyed by components of the immune system), the use of immunosuppressant drugs is indicated. However, the only presently approved treatment options for many patients are platelet transfusions and bone marrow transplantation. Prophylactic transfusions are used sparingly because they may lose their effectiveness with repeated use due to the development of platelet alloantibodies. Additional potential risks of platelet transfusion include infection, anaphylaxis, and hemolytic reactions. In platelet dysfunction or thrombocytopenia caused by decreased production, transfusions are reserved for patients with active bleeding or severe thrombocytopenia (e.g., platelet count <10,000/μL). In thrombocytopenia caused by platelet destruction, transfusions are reserved for life-threatening or CNS bleeding.
Since TPO has been shown to promote MK proliferation and maturation and platelet formation (Kaushansky et al., 1994), the use of TPO in the treatment of thrombocytopenia has been suggested. It was reported that when administered intravenously to normal healthy volunteers and cancer patients, recombinant human TPO produced a dose-dependent increase in platelet counts beginning 5 days after administration and peaking 10-14 days later. However, clinical testing of early thrombopoietin analogues was stopped because antibodies cross-reacted with endogenous thrombopoietin and caused secondary thrombocytopenia and bleeding (Junzhi et al., 2001). Under certain conditions, TPO is suggested as maintenance therapy rather than for inducing remission.
Other cytokines such as IL-1, IL-3, IL-6 and GM-CSF have been shown to play a role in the generation of megakaryocytes in animals and have demonstrated thrombopoietic activity in clinical studies. However, each either exhibits unacceptable toxicity profiles or does not produce significant increases in platelet counts, and further therapeutic use of these cytokines in the treatment of thrombocytopenia has been discontinued.
It is therefore clear that an unmet need for effective agents as an alternative to platelet transfusions that prevent and/or treat thrombocytopenia exists.
The chemokine receptor CXCR4 is a G-protein coupled receptor that is expressed in a wide assortment of normal tissues, and plays a fundamental role in fetal development, mobilization of hematopoietic stem cells and trafficking of naive lymphocytes (Rossi and Zlotnik, 2000). The chemokine CXCL12 (also known as stromal-derived factor-1, or SDF-1) is CXCR4's only natural ligand. CXCL12 is expressed constitutively in a variety of tissues, including lung, liver, bone marrow and lymph nodes.
Binding of CXCL12 to CXCR4 activates a variety of intracellular signal transduction pathways and effector molecules that regulate cell chemotaxis, adhesion, survival, and proliferation. For example, the phosphatidyl-inositol-3-kinase pathway and the mitogen-activated protein (MAP) kinase pathways are regulated by CXCL12 and CXCR4.
It has been shown that mature MKs functionally express the SDF-1 receptor, CXCR4. It was also found that SDF-1 induced the migration of mature MKs through endothelial cell layers in vitro and increased their platelet production. In addition, adeno-SDF-1 injection into normal mice resulted in increased platelet counts after 3 days, which peaked at days 7 to 10 and returned to normal by day 28 (Lane et al., 2000).
Various uses of chemokine receptor modulators, including CXCR4 agonists and antagonists, have been described in the art (Princen et al., 2005; Tamamura et al., 2005; U.S. Pat. No. 7,169,750). U.S. Pat. No. 7,435,718 discloses certain SDF-1 analog peptides that act as CXCR4 antagonists, and which may be used to treat hematopoietic cells, such as progenitor or stem cells, to promote the rate of cellular multiplication, self-renewal, proliferation or expansion. The disclosure of U.S. Pat. No. 7,435,718 suggests that these SDF-1 analogs may be formulated or administered with additional active ingredients, inter alia TPO.
US Pub. No. 2007/0167459 discloses heterocyclic compounds having CXCR4 regulating activity, in particular CXCR4 antagonists. These compounds are suggested for the prevention and treatment of various diseases, inter alia a cancerous disease including thrombocytopenia. The specification also discloses the use of these compounds with additional drugs or compounds selected from a broad list including TPO.
The bicyclam drug termed AMD3100, originally discovered as an anti-HIV compound, specifically interacts with CXCR4 in an antagonistic manner. Blocking CXCR4 receptor with AMD3100 results in the mobilization of hematopoietic progenitor cells. PCT Pub. No. WO 03/011277 is directed to a method to enhance the population of progenitor and/or stem cells in a subject by administering CXCR4 antagonists such as AMD3100, optionally with co-administered TPO. AMD3100 is undergoing clinical trials to evaluate its ability to increase stem cells available for transplant and is indicated (under the trade name Mozobil) in combination with granulocyte-colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) to the peripheral blood for collection and subsequent autologous hematopoietic stem cell transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma. However, as can be determined from the product insert of Mozobil, administration of this drug may result in reduced platelet levels and thrombocytopenia, and accordingly platelet levels must be monitored during Mozobil use and apheresis.
Thus, conflicting evidence exist as to the possible involvement of various CXCR4 agonists and antagonists in modulating platelet levels.
T-140 is a 14-residue synthetic peptide developed as a specific CXCR4 antagonist that suppress HIV-1 (X4-HIV-1) entry to T cells through specific binding to CXCR4 (Tamamura et al., 1998). Subsequently, peptide analogs of T-140 were developed as specific CXCR4 antagonist peptides with inhibitory activity at nanomolar levels (see Tamamura et al., 2003, WO 2002/020561 and WO 2004/020462).
WO 2002/020561 discloses novel peptide analogs and derivatives of T-140. The '561 publication demonstrates that the claimed peptides are potent CXCR4 inhibitors, manifesting high anti-HIV virus activity and low cytotoxicity.
WO 2004/020462 discloses additional novel peptide analogs and derivatives of T-140, including 4F-benzoyl-TN14003 (SEQ ID NO: 1). The '462 publication further discloses novel preventive and therapeutic compositions and methods of using same utilizing T-140 analogs for the treatment of cancer and chronic rheumatoid arthritis. The specification of '462 demonstrates the ability of these peptides to inhibit cancer cell migration, including breast cancer and leukemia cells, and to inhibit metastasis formation in vivo. Further demonstrated therein is inhibition of delayed-type hypersensitivity reaction in mice and collagen-induced arthritis, an animal model of rheumatoid arthritis.
WO 2004/087068 is directed to a method for treating or preventing a CXCR4 mediated pathology comprising administering a CXCR4 peptide antagonist to a host in an amount sufficient to inhibit CXCR4 signal transduction in a cell expressing a CXCR4 receptor or homologue thereof, wherein the CXCR4 peptide antagonist is not an antibody or fragment thereof. The '068 publication discloses that exemplary CXCR4 peptide antagonists include T140 and derivatives of T140, and that the pathology includes cancer such as breast, brain, pancreatic, ovarian, prostate, kidney, and non-small lunch cancer. Other publications directed to the use of CXCR4 antagonists in cancer therapy include, for example, WO 00/09152, US 2002/0156034, and WO 2004/024178.
A publication to some of the inventors of the present invention (Avniel et al., 2006) discloses that blocking the CXCR4/CXCL12 axis by a T-140 analog resulted in a significant reduction in eosinophil accumulation in the dermis and improved epithelialization, thus significantly improving skin recovery after burns.
Subsequently, it was discovered that under certain conditions, some of the functions of T-140 analogs may have CXCR4 super-agonistic properties, in addition to their CXCR4 antagonistic activity. WO 2008/075369 to some of the inventors of the present invention discloses compositions comprising T-140 peptide analogs having CXCR4 super-agonist activity and therapeutic uses thereof in modulating recovery of the hematopoietic system, particularly in the treatment of conditions associated with damage to the bone marrow. WO 2008/075370 to some of the inventors of the present invention discloses compositions comprising T-140 peptide analogs having CXCR4 super-agonist activity and therapeutic uses thereof in cancer therapy. WO 2008/075371 to some of the inventors of the present invention discloses compositions comprising T-140 peptide analogs having CXCR4 super-agonist activity and novel therapeutic uses thereof for immunotherapy and vaccination.
None of the prior art discloses or suggests that CXCR4 inhibitor peptides belonging to the T-140 analog family specifically promote the production of platelets in vivo, and particularly that these peptides may potentiate the ability of TPO to elevate platelet counts. There exists a long felt need for compositions and methods useful for treating and preventing platelet deficiencies. Therapeutic agents capable of enhancing platelet counts in an acute manner, useful for controlling bleeding in a subject in need thereof, would also be advantageous.